Matriptase (also termed epithin and MTSP-1) is a novel, epithelial cell-specific, type II transmembrane serine protease (TTSP), whose initial characterization was funded under an NIH R21 grant. We also characterized HAI-1 (hepatocyte growth factor activator inhibitor-1) as the physiologically relevant, cognate inhibitor of matriptase-the only cognate inhibitor yet described for a TTSP family member. We now know that expression of high levels of both matriptase and HAI-1 in early stage (non-metastatic) human breast cancer are significantly correlated with poor patient outcome. We hypothesize that, based on its constitutive activation in breast cancer, its important substrates (urokinase and hepatocyte growth factor), and its correlation with poor prognosis human breast cancer, that matriptase may be involved in malignant progression (metastasis) of the disease. In addition, we propose that this enzyme may be a target for therapy of human breast cancer. In our prior published work, we have shown that in non-malignant mammary epithelial cells, activation of matriptase depends upon exposure of the cells to serum, serum-derived lipopoproteins, or lipoprotein-derived sphingosine-1-phosphate. Activation of matriptase is associated with its extracellular shedding, and glycosylation of matriptase dramatically prolongs the half life of its protein and its enzymatic activity. Indeed, in our recent collaborative study, enforced expression of matriptase in gastric carcinoma cells enhanced their metastatic ability; studies in breast cancer are underway. Matriptase may be unique among carcinoma-expressed proteases, in that its expression is both restricted to the epithelial component and its activation is autonomous in cancer cells, compared to non-cancer cells. Because of its constitutive activation, directly on breast cancer cell surfaces, matriptase may function upstream of other malignancy-associated, pericellular events. Inhibition of matriptase may therefore provide a key target for interfering with the ability of carcinomas to degrade the extracellular matrix (ECM), to activate growth factors, and to promote cell motility, all cellular features of progression-associated, epithelial-mesenchymal transitions of poor prognosis carcinomas. In the current grant proposal, we will carry out 3 Aims. First, we will further elucidate the mechanisms of activation of matriptase in non-malignant, contrasted to malignant mammary epithelial cells. Second, we will examine functional aspects of matriptase and HAI-1 in human xenograft and transgenic models of mammary cancer. Finally, we will further examine the pathophysiologic roles of matriptase and HAI-1 in archival specimens of human breast cancer. These studies could lead to new perspectives on the mechanisms of regulation and cancer-associated role(s). They may also provide new avenues for diagnosis and therapy of human breast cancer.